HK1259689B - Systems and methods for improved focus tracking using blocking structures - Google Patents

Description

Systems and methods for improved focus tracking using blocking structures

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/468,355, filed March 7, 2017, which is hereby incorporated by reference in its entirety. This application also claims priority to Netherlands Patent Application No. N2018854, filed May 5, 2017, which is hereby incorporated by reference in its entirety.

background

Many advances in the biological field have benefited from improved imaging systems and techniques, such as those used in optical microscopes and scanners. Obtaining accurate focus during imaging using these imaging systems can be important for successful imaging operations. Additionally, reducing the time delay associated with focusing the system on a sample increases the speed at which the system can operate.

Many existing scanning systems use multi-beam focus tracking systems to determine the focal distance for a given sample. Multi-beam systems use an objective lens to focus two beams onto the sample. The focused beams reflect off the sample's surface, and the reflected beams are directed toward an image sensor. The reflected beams form light spots on the image sensor, and the distance between the light spots can be used to determine the focal distance.

Designers of existing systems continually strive to improve focus accuracy and the speed at which the system can determine a focus setting. Improving accuracy can be important because it allows the system to achieve better results. Reducing latency can be an important consideration because it allows the system to determine focus more quickly, thereby allowing the system to complete scanning operations more quickly.

Overview

Various examples of the technology disclosed herein provide systems and methods for improving the accuracy of focus tracking in optical systems. Additional examples provide systems and methods for reducing the latency associated with focus tracking in optical scanners. In some examples, systems and methods are provided for improving or reducing the latency associated with focus tracking in optical scanners.

In some examples, the optical blocking structure includes: a frame defining an aperture; first and second elongated structural members, each elongated structural member including a first and a second end, such that the first and second elongated structural members are connected to opposite sides of the aperture at their first ends, and further such that the first and second elongated structural members extend from the frame parallel to each other and in the same direction as each other; and an optically opaque blocking member positioned to extend between respective second ends of the first and second blocking members.

As just another example, the front surface of the blocking member may form a blocking surface sized to block a light beam reflected from a first surface of a sample container in an imaging system without blocking a light beam reflected from a second surface of the sample container. Furthermore, as just another example, the optical blocking structure may be sized to be positioned adjacent to the beam splitter, with the rear surface of the blocking member positioned in a plane angled relative to the plane of the frame, further such that the angle is selected such that the rear surface of the blocking member is substantially parallel to a plane of the beam splitter. In some instances, the width of the rear surface of the blocking member may be between approximately 2 mm and 7 mm. Furthermore, as just another example, the rear surface of the blocking member may have a width selected to block a light beam reflected from the second surface of the sample container without interfering with light beams reflected from surfaces S2 and S3. In another example, the width of the rear surface of the blocking member is selected to block a light beam reflected from the second surface of the sample container without interfering with light beams reflected from surfaces S2 and S3.

Furthermore, in another example, the optical blocking structure may be sized for use in conjunction with a scanning system that images a sample contained in a multi-layer sample container that may include four reflective surfaces, with the rear surface of the blocking member further forming a blocking surface sized to block reflected light beams from a first surface of the sample container in the imaging system without blocking reflected light beams from second and third surfaces of the sample container. As just another example, the optical blocking structure may be sized to have a triangular cross-section.

In other examples, the optical blocking structure may include a frame defining an aperture, such that the aperture is sized to be from approximately 20 mm to 40 mm in width and from approximately 15 mm to 30 mm in height; a first structural member may include a first end and a second end defining a slender body, the slender body being sized to be between approximately 20 mm and 30 mm in length; a second structural member may include a first end and a second end defining a second slender body, the slender body being sized to be between approximately 20 mm and 30 mm in length, such that the first and second structural members are connected to opposite sides of the aperture at their first ends, wherein the first and second structural members extend from the frame parallel to each other and in the same direction as each other; and an optically opaque blocking member positioned to extend between respective second ends of the first and second blocking members, the optically opaque blocking member being sized to be between approximately 1 mm and 20 mm in width.

By way of example only, a front surface of the blocking member may form a blocking surface that is dimensioned to block a reflected light beam from a first surface of a sample container in an imaging system without blocking a reflected light beam from a second surface of the sample container. Furthermore, by way of another example only, the optical blocking structure may be dimensioned to be disposed adjacent to a beam splitter, with the rear surface of the blocking member disposed in a plane that is angled relative to the plane of the frame, further wherein the angle is selected such that the rear surface of the blocking member is substantially parallel to a face of the beam splitter.

In some examples, the width of the rear surface of the blocking member can be selected to block the reflected light beam from the second surface of the sample container without interfering with the light beams reflected from the S2 and S3 surfaces. In some instances, by way of example only, the optical blocking structure can be sized for use in conjunction with a scanning system that images a sample contained in a multi-layer sample container that may include four reflective surfaces, and further such that the rear surface of the blocking member can form a blocking surface sized to block the reflected light beam from the first surface of the sample container in the imaging system without blocking the reflected light beams from the second and third surfaces of the sample container. Additionally, the optical blocking structure can be sized to have a triangular cross-section.

In some examples, an imaging system may also be disclosed, wherein the imaging system includes a laser light source; a differential separation window that separates light emitted from the light source into a plurality of parallel light beams; a sample container that may include first, second, and third layers arranged in a contacting relationship on a substrate, the layers including a first reflective surface at the first layer of the sample container, a second reflective surface between the first and second layers, a third reflective surface between the second and third layers, and a fourth reflective surface between the third layer and the substrate; an objective lens positioned to focus light from the light source onto at least one of the second and third reflective surfaces and receive light reflected from the sample container; an image sensor that includes a plurality of pixel positions positioned to receive light reflected from the sample container; a first blocking structure positioned to prevent reflections from the first surface from reaching the image sensor; and a second blocking structure positioned to prevent reflections from the fourth surface from reaching the image sensor.

As an example, the imaging system may further include a splitter arranged in an optical return path of the imaging system between the objective lens and the image sensor, further such that the second blocking structure is positioned in the return optical path to block reflections from the fourth surface at an output of the splitter.

In an additional example, the second blocking structure may further include: a frame defining the aperture; first and second elongated structural members, each elongated structural member including a first and a second end, such that the first and second elongated structural members are connected to opposite sides of the aperture at their first ends, and further such that the first and second elongated structural members extend from the frame parallel to and in the same direction as each other; and an optically opaque blocking member positioned to extend between the respective second ends of the first and second blocking members. Furthermore, in some examples, a front surface of the blocking member may form a blocking surface dimensioned to block a reflected beam from a first surface of a sample container in the imaging system without blocking a reflected beam from a second surface of the sample container. In an additional example, and by way of further example only, the second blocking structure may be dimensioned to be disposed adjacent to the beam splitter, and a rear surface of the blocking member may be disposed in a plane angled relative to the plane of the frame, further such that the angle is selected such that the rear surface of the blocking member is substantially parallel to a face of the beam splitter.

By way of example only, the width of the rear surface of the second blocking structure may be between approximately 2 mm and 7 mm. Furthermore, by way of another example only, the second blocking structure may be sized for use in conjunction with a scanning system that images a sample contained in a multi-layer sample container that may include four reflective surfaces, and further such that the rear surface of the blocking member forms a blocking surface sized to block reflected light beams from a first surface of the sample container in the imaging system without blocking reflected light beams from second and third surfaces of the sample container.

Additionally, the first blocking structure may include an aperture sized to block the reflected light beam from the first surface of the sample container and to allow the light beams reflected from the second and third surfaces of the sample container to pass through the aperture.

Other features and aspects of the disclosed examples will become apparent from the following detailed description taken in conjunction with the accompanying drawings, which illustrate, by way of example, features of examples according to the invention. This summary is not intended to limit the scope of the invention, which is defined solely by the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology disclosed herein, implemented according to one or more examples, is described in detail with reference to the following figures. These figures are provided to facilitate the reader's understanding of the disclosed technology and are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Indeed, the figures in the accompanying drawings are provided for illustrative purposes only and depict only example implementations of the disclosed technology. In addition, it should be noted that for clarity and ease of illustration, the elements in the drawings are not necessarily drawn to scale.

Some of the drawings included herein show various examples of the disclosed technology from different perspectives. Although the accompanying descriptive text may refer to such views as "top," "bottom," or "side" views, such references are merely descriptive and do not imply or require that the disclosed technology be implemented or used in a specific spatial orientation unless explicitly stated otherwise.

FIG1 shows a simplified block diagram of one example of an image scanning system with which the systems and methods disclosed herein may be implemented.

Figures 2A and 2B illustrate example optical systems for focus tracking. In particular, Figure 2A illustrates an example optical design for focus tracking according to one example implementation of the systems and methods described herein. Figure 2B is a schematic diagram illustrating an alternative view of a portion of the optical system shown in Figure 2A.

FIG. 3A illustrates an example of a sample container including multiple layers configured to hold one or more samples to be imaged.

3B is a diagram illustrating an example of the creation of desired and unwanted reflections from multiple surfaces of a multi-layered sample container in some environments.

FIG. 3C is a graph illustrating an example of the effects of unwanted reflections on an image sensor.

3D is a graph illustrating the reduction of noise at an image sensor as a result of placement of a blocking structure according to an example application of the techniques disclosed herein.

4A illustrates a portion of an image sensor including a plurality of pixels (not shown for clarity of illustration) onto which a focus tracking beam is directed, according to one exemplary implementation of the systems and methods described herein.

4B is a graph showing the intensities of left and right focused beams reflected from the S2 and S3 surfaces onto the image sensor at different focus settings using a collimating lens adjusted to position the beam waist along the optical path of the focused tracking beam.

4C is a graph showing the intensities of left and right focused beams reflected from the S2 and S3 surfaces onto the image sensor at different focus settings using a collimating lens adjusted to be more optimally positioned at the beam waist along the optical path of the focused tracking beam.

FIG. 5A is a diagram illustrating an example of a lens implemented to cause a focus tracking beam to converge on a sample plane and be focused onto an image sensor.

FIG. 5B is a diagram illustrating an example of a roof prism implemented to converge a focus tracking beam onto an image sensor.

6 is a diagram illustrating an example configuration including lenses positioned to place the beam waist of a focused tracking beam at a selected location.

7 is a diagram illustrating an example of a focus tracking system with which the systems and methods described herein may be implemented.

8 and 9 are diagrams illustrating the spatial relationship of reflected focus tracking beams in one example.

10 is a diagram showing example placement of beam blocks that block reflections of the left and right focus tracking beams from the S4 surface.

11 and 12 are diagrams showing the spatial relationship of reflected focus tracking beams at the beam splitter in the example configuration of FIG. 7 , with the beam blocker positioned as shown in FIG. 10 .

13 and 14 show light beams reflected from a top periscope and a beam splitter in one example.

15A is a top-down view showing an example of focus tracking beams reflected from surfaces S2 and S4 returning from the objective lens and directed toward the splitter.

FIG. 15B is a close-up view of FIG. 15A , illustrating how the S4 reflected beam can be blocked at the rear surface of the splitter by a blocking member.

15C is a diagram illustrating a top-down view of an example of a blocking member located at the rear surface of a separator.

15D is a diagram showing a representation of a 4 mm wide blocking structure in the beam path of the reflected focus tracking beam at the splitter.

16A and 16B are diagrams illustrating examples of beam blockers that may be used to block S4 reflections at a filter/splitter according to the example implementations described with reference to FIGs. 8-10.

17A presents a cross-sectional view of a beam block installed at a beam splitter in one example.

FIG17B presents a rear view of a beam block mounted at the beam splitter.

FIG. 18A shows an example of an aperture that may be used to block a light beam reflected from the S1 surface.

FIG18B shows an example placement of an aperture in front of a beam splitter perpendicular to the beam axis.

FIG19 shows the light spot from the top periscope for the beam focused at the top of the sample.

FIG20 shows the light spot from the top periscope for the beam focused at the bottom of the sample.

FIG. 21 shows the spots at the camera for the S2 , S3 reflected beams for imaging at the top of the capture range when focusing on S2 .

FIG. 22 shows the light spots at the camera for the S2 , S3 reflected light beams for imaging at the bottom of the capture range when focusing on S2 .

FIG. 23 shows the spots at the camera of the S2 , S3 reflected beams for imaging at the top of the capture range when focusing on S3 .

FIG. 24 shows the light spots at the camera of the S2 , S3 reflected light beams for imaging at the bottom of the capture range when focusing on S3 .

FIG. 25A shows, in one example, spot edge variation in a beam spot on an image sensor with a laser diode operating in a laser emission mode.

FIG. 25B shows a spot profile in a beam spot on an image sensor with the laser diode operating in a low power mode in one example.

FIG. 26 is a diagram illustrating an example of a laser diode operating in an ASE mode.

FIG. 27 is a diagram illustrating an example of a laser diode operating in a laser emission mode.

FIG. 28 is a diagram illustrating an example of a laser diode operating in a hybrid mode.

FIG. 29 illustrates the instability of the spot size when the laser diode is powered to operate in the lasing mode.

FIG. 30A shows an example of spot movement in a case where a laser diode operates in a hybrid mode.

FIG. 30B shows an example of spot movement in a case where the laser diode operates in hybrid mode.

FIG. 31 is a graph showing examples of spectral widths of various laser sources that were tested to determine the relationship between the spectral width at 5% and the set power.

It should be understood that the disclosed technology can be practiced with modification and alteration and that the disclosed technology is limited only by the claims and their equivalents.

Detailed description

Various example implementations of the technology disclosed herein provide systems and methods for increasing or decreasing the latency associated with focus tracking in an optical scanner. Various additional examples provide systems and methods for improving the accuracy of a focus tracking system in an optical scanner. Still other examples combine aspects of both. For example, in some examples, systems and methods are provided to prevent stray light caused by unwanted reflections from a sample container from reaching an image sensor and obstructing detection of a focus tracking beam. In some applications, a sample container for a scanning system may include a sample layer sandwiched between two or more other layers. In such applications, the surfaces presented by the multi-layered sample container may each reflect a light beam back toward the objective lens and into the return path of the scanning system. Unwanted reflections, which in some cases may be much stronger than reflections from the sample layer, can reduce the signal-to-noise ratio at the image sensor, making it difficult to detect the actual focus tracking beam amidst all the other optical noise. Unwanted reflections or scattered light beams may also overlap with and coherently interfere with the focus tracking spot at the image sensor, causing edges to appear, thereby reducing focus tracking accuracy. Examples of the systems and methods disclosed herein may place apertures, blocking bars, or other blocking members at one or more points along the return signal path to provide an optically opaque structure to block unwanted light beams reflected from other surfaces from reaching the image sensor.

As another example, additional configurations may include optical structures, such as lenses or other curved or partially curved optical elements in the optical path, to shape the focus tracking laser beam. In various examples, this may be achieved by inserting an optical element in the optical path before the objective lens to position the beam waist within the system. More specifically, in some implementations, the optical element is positioned in the optical path at a determined distance from the output end of the optical fiber so as to place the beam waist of one or more focus tracking beams at a desired location along the optical path. The position of the beam waist along the optical path may be selected so that the resulting spots of focus tracking beams reflected from multiple surfaces of interest of the sample container are the same size or substantially the same size as each other at the image sensor plane to improve focus tracking accuracy and latency. In additional implementations, an adjustment mechanism may be provided to adjust the positioning of the optical element for optimal placement of the beam waist along the optical path.

As yet another example, other implementations include configuration and adjustment of a light source for a focus tracking beam. More specifically, some examples may be configured to adjust and set the power level at which a laser diode source operates to reduce the edge flux of the focus tracking beam spot on the image sensor and provide a more stable and consistent spot placement. The power level of the laser may be set so that the laser diode operates in a quasi-laser emission mode or a hybrid mode, combining aspects of an ASE mode of operation and a laser emission mode of operation. The power level may be set within a range where the high end is lower than the power at which the laser diode emits light having a single main spectral peak and negligible side peaks, which is generally considered to be highly coherent, and the low end is higher than the power at which the laser fully emits temporarily incoherent light also known as amplified spontaneous emission (ASE).

Before describing additional examples of the systems and methods disclosed herein, it is useful to describe an example environment in which the systems and methods can be implemented. One such example environment is an environment of an image scanning system such as that shown in the figures. An example imaging scanning system may include a device for acquiring or generating an image of an area. The example outlined in FIG1 shows an example imaging configuration for a backlight design implementation.

As can be seen in the example of FIG1 , a subject sample is positioned on a sample container 110, which is located on a sample stage 170 below an objective lens 142. A light source 160 and associated optics direct a beam of light (such as a laser) to a selected sample position on the sample container 110. The sample fluoresces and the resulting light is collected by the objective lens 142 and directed to a light detector 140 to detect the fluorescence. The sample stage 170 moves relative to the objective lens 142 to place the next sample position on the sample container 110 at the focal point of the objective lens 142. Movement of the sample stage 110 relative to the objective lens 142 can be achieved by moving the sample stage itself, the objective lens, the entire optical table, or any combination of the foregoing. Additional examples may also include moving the entire imaging system over a stationary sample.

The fluid delivery module or device 100 directs the flow of reagents (e.g., fluorescent nucleotides, buffers, enzymes, lysis reagents, etc.) to (and through) a sample container 110 and a waist valve 120. In a particular example, the sample container 110 can be implemented as a flow cell comprising nucleic acid sequence clusters at multiple sample locations on the sample container 110. The sample to be sequenced can be attached to the base of the flow cell along with other optional components.

The system also includes a temperature station actuator 130 and a heater/cooler 135, which can optionally adjust the temperature of the condition of the fluid within the sample container 110. A camera system 140 can be included to monitor and track the sequencing of the sample container 110. The camera system 140 can be implemented, for example, as a CCD camera, which can interact with various filters within the filter switching assembly 145, the objective lens 142, and the focus laser/focus laser assembly 150. The camera system 140 is not limited to a CCD camera, and other camera and image sensor technologies can be used.

A light source 160 (e.g., an excitation laser within an assembly that optionally includes multiple lasers) or other light source can be included to illuminate the fluorescent sequencing reaction within the sample via illumination through a fiber optic interface 161 (which can optionally include one or more re-imaging lenses, fiber optic equipment, etc.). A low-watt lamp 165, a focusing laser 150, and an inverted dichroic mirror 185 are also presented in the illustrated example. In some examples, the focusing laser 150 can be turned off during imaging. In other examples, an optional focusing configuration can include a second focusing camera (not shown), which can be a quadrant detector, a position sensitive detector (PSD), or similar detector to measure the location of the scattered light beam reflected from the surface in parallel with data collection.

Although shown as a backlight device, other examples may include light from a laser or other light source that is directed through the objective lens 142 onto the sample on the sample container 110. The sample container 110 may ultimately be mounted on a sample stage 170 to provide movement and alignment of the sample container 110 relative to the objective lens 142. The sample stage may have one or more actuators to allow it to move in any one of three dimensions. For example, in terms of a Cartesian coordinate system, actuators may be provided to allow the stage to move in the X, Y, and Z directions relative to the objective lens. This may allow one or more sample locations on the sample container 110 to be placed in optical alignment with the objective lens 142.

In this example, a focusing (z-axis) component 175 is shown as being included to control the positioning of the optical components relative to the sample container 110 in a focusing direction (referred to as the z-axis or z-direction). The focusing component 175 may include one or more actuators physically coupled to the optical table or the sample table, or both, to move the sample container 110 on the sample table 170 relative to the optical components (e.g., the objective lens 142) to provide appropriate focus for the imaging operation. For example, the actuator may be physically coupled to the respective stage, such as by mechanical, magnetic, fluidic, or other attachment or other contact with the stage, either directly or indirectly. The one or more actuators may be configured to move the stage in the z-direction while maintaining the sample stage in the same plane (e.g., maintaining a level or horizontal attitude perpendicular to the optical axis). The one or more actuators may also be configured to tilt the stage. For example, this may be done so that the sample container 110 can be dynamically leveled to account for any tilt in its surface.

Focusing the system typically refers to aligning the focal plane of the objective with the sample to be imaged at the selected sample location. However, focusing can also refer to adjusting the system to obtain desired characteristics for the representation of the sample, such as, for example, a desired level of sharpness or contrast for the image of the test sample. Because the available depth of field of the focal plane of the objective is very small (sometimes on the order of about 1 μm or less), the focusing component 175 closely follows the surface being imaged. Because the sample container is not perfectly flat when fixed in the instrument, the focusing component 175 can be arranged to follow this profile while moving along the scan direction (referred to as the y-axis).

Light emitted from the test sample at the sample location being imaged can be directed to one or more detectors 140. The detector can include, for example, a CCD camera. An aperture can be included and positioned to allow only light emitted from the focused area to pass to the detector. The aperture can be included to improve image quality by filtering out components of light emitted from areas outside the focused area. An emission filter can be included in a filter switching assembly 145 that can be selected to record the determined emission wavelength and remove any stray laser light.

In various applications, the sample container 110 may include one or more substrates on which the sample is provided. For example, in the case of a system for analyzing a large number of different nucleic acid sequences, the sample container 110 may include one or more substrates to which the nucleic acid to be sequenced is bound, attached, or associated. In various implementations, the substrate may include any inert substrate or matrix to which the nucleic acid can be attached, such as, for example, a glass surface, a plastic surface, latex, dextran, a polystyrene surface, a polypropylene surface, a polyacrylamide gel, a gold surface, and a silicon wafer. In some applications, the substrate is within a channel or other region of a plurality of locations formed in a matrix or array throughout the sample container 110.

Although not shown, a controller may be provided to control the operation of the scanning system. The controller may be implemented to control aspects of the system operation, such as focusing, stage movement, and imaging operations. In various implementations, the controller may be implemented using hardware, machine-readable instructions, or algorithms, or a combination of the foregoing. For example, in some implementations, the controller may include one or more CPUs or processors with associated memory. As another example, the controller may include hardware or other circuitry to control operations. For example, the circuitry may include one or more of the following: a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), a programmable logic device (PLD), a complex programmable logic device (CPLD), a programmable logic array (PLA), a programmable array logic (PAL), or other similar processing devices or circuitry. As yet another example, the controller may include a combination of the circuitry and one or more processors.

Typically, for focusing operations, a focused beam generated by a focusing laser is reflected from a sample location to measure the desired focus, and the sample stage is moved relative to the optical bench to focus the optical bench on the current sample location. Movement of the sample stage relative to the optical bench for focusing is typically described as movement along the z-axis or in the z-direction. The terms "z-axis" and "z-direction" are used consistently with their common usage in the art of microscopy and imaging systems, where the z-axis refers to the focal axis. Thus, a z-axis translation results in an increase or decrease in the length of the focal axis. For example, a z-axis translation can be performed by moving the sample stage relative to the optical bench (e.g., by moving the sample stage or an optical element, or both). Thus, a z-axis translation can be performed by driving the objective lens, the optical bench, or the sample stage, or a combination of the foregoing, any of which can be driven by activating one or more servos, motors, or other actuators in functional communication with the objective lens, the sample stage, or both. In various implementations, the actuator can be configured to tilt the sample stage relative to the optical bench, for example, to effectively flatten the sample container in a plane perpendicular to the optical imaging axis. Where such dynamic tilting is performed to effectively flatten the sample position on the sample container, this can allow the sample container to be moved in the x- and y-directions for scanning as desired with little or no movement in the z-axis.

Figures 2A and 2B illustrate example optical systems for focus tracking. In particular, Figure 2A illustrates an example optical design for focus tracking. Figure 2B is a schematic diagram of an alternative view of a portion of the optical system shown in Figure 2A. To avoid clutter and facilitate the reader's understanding, the example shown in Figure 2A is shown with a single beam, which in this case is the center beam. The system can operate using more than one beam, such as, for example, with three beams. For example, a three-beam system can provide both forward-looking and backward-looking focus tracking.

Referring now to FIG2A , a laser 270 generates light for focusing the beam and is optically coupled into the system. Light from the laser 270 can be coupled, for example, via an optical fiber, to a beam splitter prism 272, such as a transversely displaced beam splitter. A filter can be included, for example, for source selection, if desired. The prism 272 splits the emitted beam into two substantially parallel spots of approximately equal intensity. This can be included to provide differential measurement in the focusing model.

The diffraction grating 274 generates multiple copies of the input beam. In other configurations, a beam splitter cube or multiple laser sources can be used to generate multiple beams. In the case of a three-beam system, the diffraction grating 274 can generate three output beams for each of the two input beams. An example for one input beam is shown in FIG2B . Because the diffraction grating can generate diverging beams (as also shown in FIG2B ), a flat-top or dove prism 276 redirects the multiple beams. In some implementations, the prism is configured so that the beams converge at the pupil of the objective lens 142 so that the beams at the sample container are perpendicular to the sample container. An example of this is shown in FIG2B for a three-output beam configuration. The received signal from the sample container returns through the beam splitter 277 and is reflected from the mirror 279. Because each beam pair diverges, the receiving prisms 280 and 282 combine the light spots onto the focal plane of the image sensor 284. In some examples, these can be implemented as dove and roof prisms to refract and collimate rays exiting the microscope object to fit onto the image sensor array. A roof prism can be used to refract the return beam to combine the spots within a spot pair onto the focal plane of the image sensor, while a dove prism refracts the front/back spot pairs to combine all spot pairs onto the focal plane. With a three-beam look-ahead, three beams pass through each of the two prism halves of the roof prism. However, on the other axis, the beams diverge, which is why a dove prism is included to correct for these issues.

In the various examples described above with reference to FIG2A and FIG2B , prisms are used to implement various optical components. Some or all of these can be implemented using lenses, however, prisms may be desirable because these components are generally less sensitive to misalignment than their lens counterparts. Prisms may also be more desirable than lens systems because they are generally more compact and include fewer elements.

The objective lens 142 in the examples of Figures 1, 2A and 2B provides a generally circular field of view on the sample container. In one implementation, the center of the field of view is the current sample position being imaged. The scanning direction within the field of view is the x or y axis. For the purposes of discussion, the direction of scanning will be assumed to be in the y direction. A light source (such as an LED or laser light source) generates a focused beam. In the example shown, three beams are used to provide a three-point differential separation axis predictive focus estimate, one beam for the current sample position and two additional beams for forward and backward focus tracking. The two additional beams are used to determine the focus distance along the z axis between the optical table and the sample position on the sample container.

The system depicted in Figures 1, 2A, and 2B illustrates an example system that the systems and methods described herein may be implemented using. While systems and methods may occasionally be described herein in the context of this example system, this is merely an example of how these systems and methods may be implemented using this example. The systems and methods described herein may be implemented using this and other scanners, microscopes, and other imaging systems.

Scanning systems that already exist use collimated light for focusing operations. In this system, collimated light of a diameter that is kept quite consistent over the entire length of the beam is directed to the objective lens. An example of this is shown in FIG. 2A (described above), in which a collimated light beam is transmitted to the objective lens. The objective lens focuses the collimated light onto the sample. The light reflected from the sample is returned by the objective lens and re-collimated. The reflected collimated light beam is then directed to the image sensor of the system (e.g., the image sensor 284 in the example of FIG. 2A ). For the purpose of focusing, the positioning of the reflected light beam on the image sensor is determined. For example, using a dual-beam system, the distance between the light spot positioning on the image sensor is measured to determine focusing.

While collimated light is a known light source and scanning system, the inventors have discovered that collimated light can adversely affect focus tracking operations in various applications. One adverse effect is related to the spot size resulting from using collimated light for the focus tracking beam. Because collimated light maintains a relatively consistent diameter throughout the optical path, the focus tracking beam images a relatively large spot size on the image sensor. This larger spot size includes a larger number of pixels on the image sensor, which increases the number of rows of image sensor pixels that need to be measured. This increases the amount of time required for the focus tracking operation.

Another adverse effect can occur in systems where the objective lens can be used to focus at multiple different surfaces but does not move by an amount equal to the distance between these different surfaces. In this case, different spot sizes of the focus tracking beam reflected from different surfaces may appear on the image sensor, affecting the focus tracking operation. Figure 3A is a diagram illustrating an example of such a phenomenon. More specifically, Figure 3A shows an example in which a sample container containing one or more samples to be imaged includes multiple layers. Now referring to Figure 3A, sample container 330 includes three layers 334, 338, and 336. In this three-layer example, there are four surfaces between the layers. These are shown at surfaces S1, S2, S3, and S4. Also shown in this example is objective lens 332, which focuses focus tracking beams 333, 335 (two in a two-beam system) onto sample container 330 for the focusing operation.

For focus tracking operations, it may be important to focus the imaging beam onto surface S2 in some instances and onto surface S3 in other instances. Assume that the separation between surfaces S2 and S3 is fixed at distance X. In some applications, depending on the operation of objective lens 332, the objective lens may be moved by a distance greater than or less than distance X when changing the focus between surfaces S2 and S3. Consequently, the focus tracking beams 333, 335 reflected from surfaces S2 and S3 may be re-collimated at different diameters, causing the S2 beam to exhibit a different spot size than the S3 beam.

An example of this is shown in FIG4A . More specifically, FIG4A shows a portion of an image sensor 362, which includes a plurality of pixels (not shown for clarity) onto which focus tracking beams are directed. On the left-hand side of the diagram in scenario 360 , this shows image sensor portion 362 with beam spots 34, 36 from each of the two focus tracking beams in a dual-beam system. Spot 34 results from left and right beams reflected from one of the two imaging surfaces (assumed to be S2 in this example), and spot 36 results from left and right beams reflected from the other of the two imaging surfaces (assumed to be S3 in this example). As shown in this example, due to the movement of the objective lens, the two focus tracking beams, both collimated and having substantially the same beam diameter before entering the objective lens, now have different diameters and therefore produce different spot sizes on the image sensor. The larger of the two spots each includes a greater number of pixels, thereby increasing the number of rows of pixels on the image sensor that need to be measured. This increases the amount of time required for the focus tracking operation. For these reasons, it is desirable to achieve a situation such as situation 361 shown on the right hand side of FIG. 4A , where the spots 34 , 36 from the left and right beams reflected from surfaces S2 and S3 , respectively, are substantially the same spot size and relatively small.

Prior art systems may use focusing lenses to converge the focus tracking beams onto the image sensor and reduce or minimize their spot size on the sensor. However, because the lenses introduce a curved surface into the optical system, slight variations in the alignment of the lenses (including variations that may be caused by thermal variations in the system) may result in inaccuracies in the placement of the focus tracking beams on the sensor. Movement or variation of the lenses may result in lateral shifts that affect the multiple focus tracking beams differently. Therefore, as described above with reference to FIG2A and FIG2B , in some examples, the focusing lenses are replaced with roof prisms.

FIG5A is a diagram illustrating an example of a focusing lens implemented to converge a focus tracking beam onto an image sensor. Referring now to FIG5A , light from a light source (e.g., laser diode 270 of FIG2A ) is transmitted to a collimating lens 400 via an optical fiber (the laser and optical fiber are not shown). The collimated light is split into two beams, such as by a beam splitter prism 382 (e.g., beam splitter prism 272 of FIG2A ). To avoid unnecessary clutter in the illustration, two reflected focus tracking beams 394, 395 are shown at lens 370 and image sensor 398; however, only one of the two focus tracking beams is shown in the remainder of FIG5A .

The focus tracking beam from beam splitter prism 382 passes through beam splitter 384 and is reflected by mirror 386 through objective lens 390. The objective lens focuses the beam onto a sample in sample container 392 (e.g., sample container 330 of FIG. 3A ). In this example, the focus tracking beam reflects from the S2 surface of sample container 392. The reflected beams (again, only one beam 394 is shown) return through objective lens 390, reflect from mirror 386 and beam splitter 384, and are directed to lens 370. Because the return beams 394, 395 diverge at this point, lens 370 is implemented to converge the return beams 394, 395 onto image sensor 398. Moreover, because the focus tracking beams 394, 395 are collimated, lens 370 provides the additional function of focusing the beams into a smaller spot size on image sensor 398. However, because changes in the lateral placement of lens 370 affect the positioning of the light beam on image sensor 398, these changes introduce focus tracking errors.

FIG5B is a diagram illustrating an example in which lens 370 is replaced with a roof prism 396 to avoid problems caused by variations in the lateral placement of lens 370. Replacing the lens with a roof prism 396 reduces or eliminates the system's sensitivity to the lateral position of the lens. Variations in the prism due to thermal and other variations do not affect the spacing of the focus tracking beams 394, 395 on image sensor 398. Since the angular deviation of the prism is entirely determined by the angle of the glass, lateral displacement of roof prism 396 does not affect the beams.

Including a roof prism 396 in place of lens 370 can improve the accuracy of the focus tracking system. Because the spacing between the light spots is used to measure the distance from the objective lens to the sample container, a higher level of accuracy is achieved when the separation of the beams depends solely on the distance to the sample container. Other variables that affect the separation of the beams (such as those introduced by lateral inaccuracies in the placement of lens 370) negatively impact the accuracy of the focus tracking system. Therefore, including a roof prism (which presents the same angle to the focus tracking beam even in the presence of some lateral displacement) can greatly benefit the accuracy of the system.

There is a disadvantage to removing the lens. Because the lens is eliminated, the focus tracking beam (in this example, beams 394, 395) is not focused on the sensor. Therefore, in various examples, rather than using collimated light as is done in preexisting scanning systems, the focus tracking beam is focused to place the waist at a given point along the optical path. This presents a smaller spot size on the image sensor. For example, in one application, the collimating lens 400 is moved further from the output end of the optical fiber than it would otherwise be placed to collimate the light from the optical fiber. The point along the optical path at which the collimating lens 400 is placed dictates where the beam waist is placed along the optical path. The collimating lens 400 can be positioned to provide a waist so that the reflected focus tracking beams 394, 395 can be focused onto the image sensor 398 with a reduced spot size despite the use of the roof prism 396 in place of the lens 370.

Another benefit of moving the collimating lens 400 to position the beam waist in the optical path is that it can help reduce or eliminate the imbalance in spot size discussed above with reference to FIG4A. The lens 400 can be provided and positioned in the optical path so that light returning from the sample through the objective lens and through the rest of the optical path impinges on the sensor with substantially the same spot size as shown in scenario 361. More specifically, in some examples, the lens is positioned at a distance from the output end of the optical fiber to place the beam waist at a predetermined distance from the collimator to balance the diameter of the light beam propagating from the upper and lower surfaces of the sample container to the image sensor.

In one application, the beam waist is located at a distance of 690mm-700mm from the collimator to balance and reduce the diameter of the beam impinging on the image sensor. In some examples, the spot size can be reduced to approximately 400μm. In other examples, the spot size can be in the range of 300μm to 500μm. In still other examples, other spot sizes can be used.

In addition, the movement of the collimating lens 400 to position the beam waist in the optical path can help balance the intensity of the light impinging on the image sensor. Figure 4B is a graph showing the intensity of the left and right focused beams reflected from the S2 and S3 surfaces onto the image sensor at different focus settings using a collimating lens adjusted to provide a beam waist at a non-optimal position. In this graph, the spot brightness is on the vertical axis and the position of the focusing stage is on the horizontal axis. In one example implementation, the vertical blue line on the left-hand side of the graph shows the optimal focus position for the S2 reflection. Similarly, in this example implementation, the vertical blue line on the right-hand side of the graph shows the optimal focus position for the S3 reflection. As shown in the graph, at the S2 focus position, the average spot brightness for the S2 beam is approximately 170, while at the optimized S3 focus position, the average spot brightness for the S3 beam is approximately 85. Therefore, the spot intensities for the S2 and S3 beams are unbalanced.

FIG4C is a graph showing the intensity of the left and right focused beams reflected from the S2 and S3 surfaces onto the image sensor at different focus settings using a collimating lens adjusted to be more optimally positioned at the beam waist along the optical path of the focus tracking beam. Here, when the beam waist is positioned along the optical path, the intensities of the S2 and S3 reflected beams are more balanced. In particular, the graph shows that at the S2 optimal focus position, the left and right S2 beams have an average spot brightness of approximately 125. This also shows that at the S3 optimal focus position, the left and right S3 beams have an average spot brightness of approximately 130. As a comparison of FIG4B and FIG4C, it is shown that the placement of the beam waist along the optical path can affect the balance of the intensities of the S2 and S3 focus tracking beams.

FIG6 is a diagram illustrating an example configuration including a lens positioned to position the beam waist of a focused tracking beam at a selected location. In this example, light from a light source (not shown), such as a laser light source (e.g., light source 270), is carried by a fiber optic cable 432, which is connected to a lens housing assembly via a ferrule 434. Ferrule 434 is mounted in a mounting block 435, which is fixedly attached to an insert 436. A lens 440 of a given focal length is positioned at a determined distance from the output end of optical fiber 432 and can be maintained at that distance by the lens housing assembly. In this example, light from the optical fiber travels through an aperture in insert 436, which is mounted in a body portion 438. The focal length of lens 440 and its distance from the output end of optical fiber 432 are selected to position the beam waist at a desired location along the optical path. As mentioned above, the distance between the output end of the optical fiber and lens 440 is selected to position the beam waist at a desired location, as described more fully below.

In this example, the spacing between lens 440 and the fiber output end is 14.23 mm, which is the working distance between the lens surface and the fiber. 15.7 mm is the effective focal length of the lens (which is higher than the back focal length of the lens because it is relative to the lens principal plane). Because the back focal length of the lens in the collimator is 13.98 mm, which is the distance from the lens vertex to the lens focus on the optical axis, the back focal length is shorter than 14.23 mm.

In the example shown, insert 436 is slidably mounted within a cavity defined by body portion 438, such that the distance between the optical fiber output end and lens 440 can be adjusted by insert 436 slidably mounted within the cavity of body portion 438. A set screw 442 or other locking mechanism can be included to lock insert 436 in place within body portion 438. The use of a slidable insert 436 allows the system to be adjusted to adjust or optimize the spot size on the image sensor. This can allow for final system configuration adjustments or field adjustments. In the example shown in FIG6 , lens 440 is a plano-convex lens. However, after reading this description, one of ordinary skill in the art will understand that other lens configurations can be used, including, for example, biconvex lenses.

In some applications, the lens is configured so that the beam waist is located within the objective. More specifically, in some applications, the lens is configured so that the beam waist is positioned within the objective before the beam impinges on the sample, while in other applications, the lens is configured so that the beam waist is positioned within the objective after the beam reflects from the sample. In other applications, the lens is configured so that the beam waist appears before the objective, after the reflected beam leaves the objective, or between the objective and the sample. The placement of the lens can be determined through an iterative process, such as through the use of modeling software, to achieve the desired spot size and balance on the image sensor.

In addition to balancing the spot size, a smaller spot size is often used to increase the speed at which focus can be determined. The time required to read information from the image sensor affects the latency of the focus tracking system. More specifically, for a sensor with a given pixel density, a larger spot size covers more pixels and requires more time to read data from each pixel within the spot diameter. Therefore, as discussed above, the lens used to balance the beam diameter can also be used to reduce the size of the spot that impinges on the image sensor, thereby reducing the amount of time required to determine the spot position (or position for multi-beam focusing) for the focusing operation.

As discussed above with reference to FIG3A , in some applications, a multi-layer sample container can be used to carry a sample to be imaged by a scanning system. As discussed in that example, the sample to be imaged can be contained in a solution in layer 338. In order for imaging to occur, at least layer 334 must be optically transparent to the light beam used for imaging. Layer 336 can also be optically transparent. Therefore, surfaces S1, S2, S3, and S4 are typically reflective. Likewise, because it is important that the imaging beam reaches the sample at layer 338, it is undesirable to use an anti-reflective coating on the surface. Therefore, unwanted reflections from surfaces S1 and S4 during focus tracking and imaging operations can generate unwanted optical noise in the system and can blur the reflected beams from S2 and S3, which are the beams collected at the image sensor.

FIG3B is a diagram illustrating an example of unwanted reflections generated off multiple surfaces of a multi-layer sample container in some environments. As seen in this example, the three-layer sample container includes surfaces S1, S2, S3, and S4. For clarity, a single focus tracking beam 465 is shown. However, in other applications, multiple focus tracking beams may be used. For example, the following example describes a system in which two focus tracking beams are described. As also seen in this example, the beams reflect off each of surfaces S1, S2, S3, and S4. Since the sample is between surfaces S2 and S3, these are the surfaces on which the system is designed to focus. Therefore, reflected beam 467 off surface S1 and reflected beam 469 off surface S4 do not return any useful information and are unwanted reflections. The reflections of interest for focus tracking are those off surfaces S2 and S3. Accordingly, if light from reflections off surfaces S1 and S4 reaches a detector, this may introduce noise that could interfere with the detection of the focus tracking beam reflections.

FIG3C is a graph illustrating an example of the effects of unwanted reflections on an image sensor. As can be seen in this example, in addition to the spot 482 presented by the focus tracking beam, there is also a significant amount of noise appearing on the image sensor as a result of the unwanted reflections. In other examples, unwanted reflections may also appear as additional spots on the image sensor. FIG3D is a graph illustrating the reduction in noise at the image sensor as a result of the placement of a blocking structure, according to an example discussed below.

This problem is exacerbated in cases where the reflections leaving surfaces S1 and S4 have a greater intensity than the reflections leaving the sample. Because it is important that the sample container be optically transparent, no anti-reflective coating is provided on the sample container. Similarly, reflections leaving glass surfaces tend to be stronger than reflections leaving biological samples. In addition, in applications where the sample container includes nanowells or other similar patterns on surfaces S2 and S3, this can further reduce reflections leaving these surfaces. Therefore, unwanted reflections from surfaces S1 and S4 tend to have a greater intensity than reflections leaving surfaces S2 and S3. For example, in some applications, reflections leaving surface S1 can be as much as 100 times (or more) the intensity of reflections leaving surfaces S2 and S3. To remedy this problem and remove the effects of these unwanted reflections from focus tracking operations, various examples can be implemented to include blocking structures at determined locations along the optical path between the sample and the image sensor to prevent this unwanted light from reaching the image sensor.

FIG7 is a diagram illustrating another example of a scanning system with which the systems and methods described herein can be implemented. Referring now to FIG7 , this example includes a light source (not shown), such as a laser light source. For example, in one application, the light source can be configured as a laser diode coupled to the system using a fiber coupler and a lens structure, such as the example shown in FIG6 . As another example, the light source can be configured as a laser diode with a collimator to provide collimated light for focus tracking operations.

In this example, light from a laser is directed into a lateral displacement prism 522 to split the light into two parallel beams. Other configurations can be implemented with a single focus tracking beam or with more than two focus tracking beams. In operation, the focus tracking beam is sent through a beam splitter 524 and reflected from an upper periscope 526 and a lower periscope 528. The focus tracking beam is transmitted through a periscope window 530 and a beam splitter 532 (which can also be implemented as a dichroic filter). The beam is then reflected from a reflector 536 and focused onto a sample container 540 by an objective lens 538. Reflections from the sample container return through the objective lens and follow the same path until they are reflected from beam splitter 524. Because the beams can diverge slightly from each other, a roof prism 546 can be included to redirect the beams into a parallel orientation or even a slightly converging configuration so that they can all be directed onto a relatively small area image sensor. In this example, a camera steering mirror 548 directs the focus tracking beam to an image sensor 550. Although the example blocking structures described herein are described in terms of this example configuration, a person of ordinary skill in the art, after reading this description, will recognize how different geometries or placements of blocking structures can be used in differently configured systems to block unwanted reflections from multi-surface sample containers.

The example system of Figure 7 was modeled to determine the paths of light beams reflected from surfaces S1-S4 in the system, identifying points along the return path where unwanted reflections from surfaces S1 and S4 could be blocked from reaching the image sensor. As a result of this modeling, the spatial relationships of the light beams at various points along the path are illustrated in Figures 8, 9, 11, 12, 19, 20, 21, 22, 23, and 24. As shown in these figures, the spatial relationships of the light beams reflected from surfaces S1-S4 vary throughout the return path of the system. The positioning of the beams varies relative to each other along the length of their return path, and also depends on the placement of the sample container relative to the objective lens. Adding to the complexity is the presence of a focusing beam extending in both the forward and return directions, as well as an imaging beam extending in both directions. Therefore, configuring blocking structures at appropriate locations within the optical path that effectively prevent unwanted reflections from transmitting noise to the image sensor while avoiding interference with the desired focus tracking and imaging beams is a nontrivial task.

FIG8 and FIG9 are diagrams illustrating the spatial relationship of the reflected focus tracking beams at beam splitter 532 in the example configuration of FIG7 using a multi-layer sample container, such as the multi-layer sample container shown in FIG3B. FIG8 and FIG9 show the beams within a 21 mm x 21 mm area. FIG8 illustrates the spatial relationship of the beams at beam splitter 532 when the system is configured to focus on the top of the sample well at surface S2, while FIG9 illustrates the spatial relationship of the beams at beam splitter 532 when the system is configured to focus on the bottom of the sample well at surface S3. These diagrams show that at beam splitter 532, when the system is focused on S2 and S3, the reflected beams impinge on the surfaces in three spatial groups: reflections of the left focus tracking beam from surfaces S1, S2, and S3 are in a first group; reflections of the right focus tracking beam from surfaces S1, S2, and S3 are in a second group physically separate from the first group; and the left and right focus tracking beams reflected from surface S4 are in the region between these two groups. With this spatial relationship among the beams, it is difficult to use an aperture configuration to effectively block the left and right reflections of surface S1 while allowing the desired reflections off surfaces S2 and S3 to pass uninhibited. However, because there is good spatial separation of the reflection off surface S4 relative to the other reflections, the reflection from the S4 surface can be blocked at this point along the return path.

FIG10 is a diagram illustrating an example placement of a beam blocker to block reflections of the left and right focus tracking beams from the S4 surface, according to one example implementation. This example shows that reflections 424 from surface S4 converge at beam splitter 532, as seen in FIG8 and FIG9. This example also illustrates how a blocking structure can be included to block these reflections from surface S4 without interfering with the desired reflections from the S2 and S3 surfaces. This can be achieved in the example shown using a 4 mm wide mask on the focus tracking module side of beam splitter 532.

Figures 11 and 12 are diagrams illustrating the spatial relationship of the reflected focus tracking beams at beam splitter 532 in the example configuration of Figure 7 using a multi-layer sample container, such as that shown in Figure 3B . Figures 11 and 12 show the beams within a 25 mm x 25 mm area. Figure 11 shows the spatial relationship of the beams at top periscope 526 when the system is configured to focus on the top of the sample well at surface S2, while Figure 12 shows the spatial relationship of the beams at top periscope 526 when the system is configured to focus on the bottom of the sample well at surface S3. Because the reflection of the focus tracking beam exiting surface S4 in this example is blocked at beam splitter 532 before reaching this point in the return path, there is no spot of light from surface S4. More importantly, this demonstrates that the reflected beam from surface S1 has good spatial separation from the desired reflections from surfaces S2 and S3.

Using this spatial placement of the beams, an aperture can be used to block the S1 reflection while allowing the reflected beams from the S2 and S3 surfaces to pass through and ultimately reach the image sensor. Figures 13 and 14 illustrate the beams reflected from the top periscope 526 and beam splitter 524. As shown, if the beams were not blocked at the top periscope 526, they would reflect from the beam splitter 524 and impinge on the edge of the roof prism 546. As shown in this modeling, the reflected beams from surface S1 can be blocked by placing a 20 mm x 20 mm aperture at the upper periscope 526. Alternatively, the size of the upper periscope 526 can be reduced to 20 mm x 20 mm so that the reflected beams from the S1 surface do not return to the image sensor. In other applications or for other placements of the aperture, the size of the aperture implemented can vary based on the location of the S1 beam. In another example implementation, the aperture is 20.8 mm wide. This width is chosen to accommodate S2 images at approximately -20 μm to +30 μm (approximately the best focus for S2 in one application) and S3 images at approximately -25 μm to +25 μm (approximately the best focus for S3 in one application).

FIG15A provides a top-down view showing a focus tracking beam reflected from a sample by an objective lens 538 and directed toward a beam splitter 532. Although mirror 536 is not shown in FIG15A , this shows the reflected focus tracking beam being reflected toward the beam splitter 532. This example also shows that the S4 reflected beam is blocked by a beam blocker located at the back of the beam splitter 532. Although a beam blocker is not shown in FIG15A , an illustrative example is provided in FIG16A and FIG16B .

FIG15B provides a close-up view of FIG15A , illustrating an example of a focus tracking beam reflected from surface S4 at the back side of beam splitter 532. As shown in this example, the focus tracking beam reflected from surface S4 is blocked by blocking member 562. As also shown in this example, the front side of blocking member 562 is oriented substantially parallel to the back side of beam splitter 532. In one example implementation, blocking member 562 is arranged in the system to be separated from the back side of beam splitter 532 by 50 μm. In other examples, other separation spacings may be provided. For example, in some implementations, the spacing may be in the range of 25 μm-100 μm. Although this example illustrates blocking member 562 as having a rectangular cross-section, blocking member 562 may be implemented using other shapes or geometries, examples of which are shown below with reference to FIG16A and FIG16B .

FIG15C is a top-down view showing an example of a blocking member and a splitter positioned within a portion of an imaging system. In this example, a blocking member 562 is positioned behind the beam splitter 532 to block the light beam reflected from the S4 surface. The reflected light beam emitted from the objective lens 538 is reflected by the mirror 536 toward the beam splitter 532. The blocking member 562 is positioned to block the light beam reflected from the S4 surface and has a width that is small enough so as not to interfere with the light beams reflected from the S2 and S3 surfaces.

In the example shown, the blocking member 562 is 4 mm wide and 2 mm long and is slightly offset from the optical axis of the objective lens 538. However, it is aligned with the center of the lower periscope 528 mounted in the housing 565. More specifically, in one example implementation, the blocking member 562 is offset 1.1 mm to the left of the objective lens optical axis to ensure that it is centered with respect to the light beam reflected from the S4 surface.

FIG15D is a diagram showing a representation of a 4 mm wide blocking structure in the beam path of a focus tracking beam reflected at a beam splitter. As shown in this example, the 4 mm wide blocking structure (represented by rectangle 631) has sufficient width to block the focus tracking beam reflected from surface S4, which is shown in the center of the diagram. As also shown in this example, the width of the blocking member is selected to be wide enough to block unwanted reflected beams, but still provide the maximum possible capture range for imaging S2 and S3. Because small changes in focus can have corresponding changes in the position of the beams at the beam splitter, the width of the blocking member can be selected to be slightly wider than necessary to block the beams under perfect focus conditions. In other words, the blocking member can be wide enough to accommodate a determined degree of inaccuracy in the focusing system.

Figures 16A and 16B illustrate examples of a beam blocker that can be used to block S4 reflections at beam splitter 532, according to the example implementation described with reference to Figures 8-10. Figures 17A through 18B illustrate exemplary placement of the beam blocker shown in Figures 16A and 16B. The left-hand side of Figure 16A shows a rear view of beam blocker 620 (as viewed from the perspective of the beam); the right-hand side shows a perspective view of beam blocker 620. Beam blocker 620 includes a frame portion 622 defining an aperture 624 through which a reflected beam can pass. A beam blocking member 626, including a blocking surface 630, is supported in position by an extension arm 628 to block unwanted reflected beams from S4. In the example shown, extension arm 628 is an elongated structural member attached, fixed, joined, or otherwise connected to opposite sides of frame portion 622, with beam blocking member 626 extending beyond the distal end of extension arm 628.

The frame portion 622 and the extension arm 628 provide a mounting structure by which the beam block member 626 can be mounted in position at the beam splitter 532 without interfering with reflections from surfaces S2 and S3. The beam block 620 can be cast, molded, machined, or otherwise manufactured as a unitary structure. In other examples, the elements comprising the beam block 620 can be separate components that are attached, bonded, fastened, or otherwise connected together to form a resulting assembly. The beam block 620 can be implemented using a light-absorbing opaque surface to prevent other unwanted reflections within the system. For example, the beam block 620 can be made of black anodized aluminum or other light-absorbing or light-absorbing coating material. The beam block 620 can be sized for a specific application. In one example application, the beam block 620 is sized to provide: an aperture width of 30 mm and a height of 21 mm; an extension arm 628 of 25 mm length; and a blocking surface of 2.8 mm wide and 21 mm long.

Referring now to FIG16B , view 682 shows a top-down view of beam blocker 620, and view 683 shows a cross-sectional side view at point A of beam blocker 620. The leading edge of extension arm 628 tapers to conform to the angle of beam splitter 532, as further illustrated in FIG17A and FIG17B (described below). The beam blocking member has a triangular cross-section and is oriented to present a flat blocking surface 630 to the incident beam. While light-absorbing materials can be used to fabricate beam blocker 620, presenting a triangular cross-section to the unwanted beam can have the effect of reflecting any unabsorbed light from the return path.

FIG17A shows a cross-sectional view of beam blocker 620 mounted at beam splitter 532. Referring now to FIG17A, in operation, reflections of the focus tracking beam from surfaces S1, S2, S3, and S4 travel upward from the objective lens, reflect from mirror 536, and are directed toward beam splitter 532. Blocking surface 630 of blocking member 626 (see FIG16A and FIG16B) prevents the S4 reflection from continuing through beam splitter 532. In the example shown, extension arm 628 is sized to position blocking member 626 at or near a surface of beam splitter 532. The figure also shows the tapered front angle of extension arm 628 to allow blocking surface 630 of blocking member 626 to be positioned adjacent to and at substantially the same angle as beam splitter 532. In some examples, blocking member 626 is positioned so that blocking surface 630 is in contact with beam splitter 532. In other examples, the blocking member 626 is positioned so that the blocking surface 630 is separated from the surface of the beam splitter 532 by a small amount, such as, for example, 50 μm to 500 μm.

In alternative examples, a blocking element may be disposed on the back side of the beam splitter 532 without the structures shown in Figures 16A to 17B. For example, in some examples, a strip of opaque material may be attached to the back side of the beam splitter 532. In other examples, an opaque or optically absorptive coating may be applied to the rear portion of the beam splitter 532 in narrow stripes.

For scanning operation, imaging beams (which can be, for example, red and green imaging beams) enter the system from the right-hand side, as indicated by arrows 690. These beams are reflected from the front face of the beam splitter 532 toward the mirror 536. The mirror 536 reflects the imaging beams downward into the objective lens. Therefore, the position of the blocking member 626 is also selected so as not to interfere with the imaging beams reflected toward the sample (through the front face of the beam splitter 532).

This example also shows that the blocking member 626 presents a triangular cross-section, wherein the rear edge of the blocking member 626 tapers to meet at an acute angle. Other cross-sectional geometries for the blocking member 626 may be used, assuming that the blocking surface 630 is appropriately sized to block or substantially block reflections from the surface S4. However, a geometry that decreases in cross-section toward the rear of the blocking member 626, such as the geometry shown, may minimize the chance that the blocking member 626 may otherwise provide unwanted interference with the desired light beam.

17B presents a rear view of the beam blocker 620 mounted at the beam splitter 532. This shows the frame portion 622 mounted in place using bolts 732. This shows the window provided by aperture 624, which allows light reflected from surfaces S2 and S3 (and S1 later in the path to be blocked) to pass through, while the blocking member 626 blocks light from surface S4 before it exits the beam splitter 532.

FIG18A shows an example of an aperture that can be used to block beams reflected from the S1 surface. In one example, this can be placed on the inner wall of the focus tracking module at the periscope aperture. As mentioned above, in one example implementation, the aperture is 20.8 mm x 20.8 mm, but other aperture sizes can be provided in other examples. As with the blocking member, the size of the aperture can be selected to block unwanted reflections while providing the maximum possible capture range of the S2 and S3 reflected beams relative to "best focus" considerations. FIG18B shows an example placement of the aperture 740 in front of the beam splitter 524 perpendicular to the beam axis.

Figures 19 and 20 show the results of the additional beam blocker 620 that blocks the S4 reflection and the 20.8 mm x 20.8 mm aperture that blocks the S1 reflection. Figure 19 shows the spot of the beam from the top periscope 526 for focusing at the top of the sample (surface S2), and Figure 20 shows the spot of the beam from the top periscope 526 for focusing at the bottom of the sample (surface S3).

While the above is illustrated using an objective lens focused at surfaces S2 and S3, perfect focus is not always achieved, and therefore examples can be implemented to account for the capture range above and below the upper and lower samples. For example, the above modeling was also performed assuming a "best focus" adapted to focus within +/- 25 μm from the upper and lower sample surfaces. This "best focus" modeling confirmed that the above configuration was sufficient to block unwanted reflections from the S1 and S4 surfaces under best focus operation.

Figures 21-24 are diagrams showing the placement of light spots at the image sensor at the top and bottom of an example "best focus" capture range. In this example, modeling was performed with a capture range of +/- 25 μm. These figures show an image sensor area of 11.26 mm x 11.26 mm. Figure 21 shows the light spots at the camera for the S2 and S3 reflected light beams used for imaging at the top of the capture range for focusing on S2 using an objective lens position of 1.064 mm from S2. Figure 22 shows the light spots at the camera for the S2 and S3 reflected light beams used for imaging at the bottom of the capture range for focusing on S2 using an objective lens position of 1.014 mm from S2. Figures 21 and 22 show variations of +/- 25 μm from the ideal focus position. Figure 23 shows the light spots at the camera for the S2 and S3 reflected light beams used for imaging at the top of the capture range when focusing on S3. FIG. 24 shows the light spots at the camera for the S2 , S3 reflected light beams for imaging at the bottom of the capture range when focusing on S3 .

As described above, in focus tracking operations utilizing a multi-beam system, the spot separation, or distance, between the spots of the focus tracking beam on the image sensor is measured to determine focus. Therefore, the stability of the spot separation can be an important factor in achieving accurate measurements. Spot separation stability can be affected by factors such as the movement of the spot quality/shape during the focus stage (sometimes referred to as the Z stage) as a function of time, and the resolution of the centroid algorithm used to resolve the spot separation. One challenge to spot separation stability is that the spot inherently includes edges. Due to mode hopping of the laser, the edge pattern can change, which causes changes in the spot profile over time, which affects the spot separation stability of the focus tracking module. An example of this is shown in FIG25A , which illustrates spot edge variation. This example shows the spot edge variation of a laser operating at a power of 12 mW, with an exposure time of 250 μs, and using an OD 1.0 ND filter in place.

Operating a laser in a mode commonly referred to as amplified spontaneous emission (ASE) tends to provide a cleaner spot profile. An example of this is shown in Figure 25B. This example is for the same laser diode operated at 500μW, 250us exposure (no ND filter). In this mode, the source emits temporally incoherent light, behaves more like an LED than a laser, and has a wide optical bandwidth of 5 to 10nm FWHM (full width at half maximum intensity). However, there are several disadvantages to operating in ASE mode, which is why generally existing imaging systems do not operate in this mode. First, ASE mode is not a lasing mode for a laser diode, so the output power is very low. It is generally defined as a mode below the lasing threshold in which no lasing occurs. Therefore, its output is temporally incoherent and includes frequency components across a wide spectrum.

FIG26 is a diagram showing an example of a laser diode operated in ASE mode. In this example, the laser diode is operated at 0.17 mW and exhibits a relatively flat spectrum (when compared to a diode operated in laser emission mode) with frequency components spanning a wide range of wavelengths. There is no single mode of operation, and the output is incoherent. Incoherence in the light source can lead to undesirable effects such as destructive interference and chromatic aberration. Additionally, operation in ASE mode may simply be impractical because there is not enough power being emitted to produce a beam of sufficient intensity. However, there are other applications in which a laser can be operated in ASE mode. In this mode, the laser diode tends to behave more like an LED, and therefore it may be useful for certain applications.

FIG. 27 is a diagram showing an example of the same laser diode operating in a laser emission mode.

The graph in the upper half of Figure 27 shows the same laser diode operating at 0.96 mW, and the graph in the lower half of Figure 27 shows the same laser diode operating at 1.22 mW. In both cases, the output is effectively highly coherent, with a single main peak at the operating frequency and almost negligible secondary peaks. This is in stark contrast to the ASE mode, which has no main peak.

Figure 28 is a graph illustrating an example of a laser diode operating in hybrid mode. Figure 28 shows the laser in this example operating at 0.43 mW. At this power level, several primary peaks begin to form, but there are still strong secondary peaks. As shown in this figure, the laser diode is not in strong lasing mode, but it is also not in full ASE mode. The power level may still be defined as above the lasing threshold, but the output is not fully coherent.

Since ASE mode may not produce output at sufficient power, operation in ASE mode is operationally impractical. As mentioned above with reference to FIG25A, however, operating the scanning system in laser emission mode produces temporally varying edges that provide instabilities in the spot measurement.

An example of this is shown in FIG29, which illustrates instabilities in the morphology of the light spot when the laser diode is powered to operate in laser emission mode according to an example of the systems and methods described herein. As shown in the figure, the standard deviation of the left beam spot on the image sensor is 1.619 pixels, while the standard deviation of the right beam spot on the image sensor is 0.518 pixels. However, as shown in the graphs for the left and right spots, the movement of the spot for each beam can be significant from one frame to the next and may actually move by several pixels. The beam profiles for two adjacent frames for the left spot are shown in the cross-sectional images on the right-hand side of the figure. These cross-sectional images illustrate how deviations in the placement of the beam spots can occur over time.

Since focus is determined by measuring the distance between the left and right light spots on the image sensor, variations in light spot placement can lead to inaccuracies in focus tracking. The effect of the motion of the left and right light beams, as shown in the top two graphs of FIG29 , is shown in the bottom graph of the figure. The graph shows the variation in the distance between the left and right light spots (referred to herein as Delta X) over the same number of frames. This shows a standard deviation of 1.178 pixels, which results in a light spot spacing stability of +/- 139 nm with a 95% confidence interval (~2*StDev for a Gaussian population). This is calculated as (1.178*1.96)/16.36=+/- 139 nm as shown in the figure. The 16.36 factor represents the focus tracking gain in pixels/μm. It indicates how many pixels of light spot spacing are obtained for every 1 μm of movement of the objective lens to the sample distance. It is used to convert increments in the light spot spacing (pixels) to increments in space in the z direction (nm).

The present inventors have discovered that interference fringe patterns appear due to the multi-level structure of the sample container, as shown in FIG3A . The present inventors have also discovered that this is the result of the superposition of multiple light beams and/or scattered light within the multi-level glass sample container. A change in the position of the sample container (e.g., in the X and Y directions) without other changes can cause the fringe to shift.

Figures 30A and 30B show additional examples of spot movement when the laser diode is operating in hybrid mode. Specifically, Figures 30A and 30B illustrate a more optimized scenario with a stable laser that is not mode-hopping. As shown in Figure 30A, the standard deviation of the left spot drops to 0.069 pixels, while the right spot drops to 0.064 pixels. As shown in the upper two graphs in the figure, the spot movement from one frame to the next is typically less than one pixel. Because the motion can be additive, the Delta X difference between the left and right spots can have a standard deviation of 0.122 pixels. This reduces the spot spacing stability to +/- 15.2 nm ((0.122 * 1.96) / 16.36 nm = +/- 15.2 nm). Here, 16.36 is the FTM gain in pixels/μm. This is the amount of Delta X in a pixel achieved when the objective lens moves 1 μm in the Z axis. This can be used to convert pixels in Delta X to μm in Z space and vice versa. Additionally, 1.96 is a multiplication factor for the standard deviation to express a 95% confidence interval for the error of the distribution (assuming it is Gaussian).

In the example of FIG30B , the standard deviation of the left spot has dropped to 0.069 pixels, and the right spot has also dropped to 0.069 pixels. As shown in the upper two graphs on the graph, the spot movement from one frame to another is typically less than one pixel. Since motion can be additive, the Delta X difference between the left and right spots may have a standard deviation of 0.127 pixels. This reduces the spot spacing stability to +/- 14.6 nm ((0.127 * 1.96) / 16.36 nm = +/- 14.6 nm).

As mentioned above, operating a laser in the previously existing ASE mode is impractical. As also just described, using a laser diode operated at power levels above the lasing threshold, accuracy suffers, and this is particularly true if mode hopping occurs (e.g., such as via power variations). However, the inventors have discovered that operating the laser in a hybrid mode between ASE mode and full lasing mode provides sufficient beam intensity for measurements at the image sensor and increased spot placement stability for improved measurement accuracy. Such a mode can be achieved in some instances by operating slightly above the lasing threshold of the laser diode. For example, this may occur slightly beyond the inflection point of the lasing curve, but still low enough that a significant portion of the power is in the ASE state. This produces an output in which a significant amount of the light still has a wider spectral width, resulting in significantly reduced coherence.

Operating the laser in this hybrid mode can be advantageous compared to other light sources that might be used to attempt to achieve the same effect. Laser diodes are often ideal light sources because they exhibit high reliability and low cost due to the high-volume manufacturing of this type of device by various companies in the field. Operating the laser diode in this lower power mode can even improve the typically high MTBF ratings achievable with laser diodes. Thus, it is possible to achieve a device with very high lifetime and MTBF ratings (a combination of laser diode characteristics and very low operating power), low manufacturing cost, and a coherence length short enough to eliminate interference fringes caused by the multi-layer structure of the sample container.

Table 1 is a graph illustrating the spot spacing stability when various optional solutions are implemented. The first set of measurements assumes a laser power of 12 mW operating in laser emission mode, the presence of an ND filter to attenuate the light, and a 250 μs exposure. Here, the center of mass or spot spacing stability is 396.02 nm for noise floor 1 and 146.0 nm for noise floor 2. As shown in the table, stability improves if 2D or 1D Gaussian filtering is added. Gaussian filters can be added to mitigate the effects of edges and provide a more uniform spot profile. As also shown in this table, reducing the power of the laser diode to 0.5 mW reduces the center of mass error, which means greater stability. In particular, in this example, the center of mass error is reduced to 14.6 nm for noise floor 1 and 15.2 nm for noise floor 2.

Table 1

In this example, operating the laser diode at 0.5 mW instead of 12 mW means that the laser is not truly in lasing mode. However, this power level is high enough that the laser diode is not operating in ASE mode either. Instead, in this power range, the laser can be said to be operating in a hybrid mode or quasi-lasing mode. This is quite unusual for laser operation. Under normal circumstances, it is expected to operate a laser in a clearly identifiable lasing mode, and pre-existing systems are comfortable operating laser diodes and power levels above the lasing threshold. Operating the laser in this hybrid mode is counterintuitive and atypical for laser operation.

FIG31 is a graph showing an example of the relationship between the 5% full spectral width (FW at 5%) and the laser power for various laser light sources. As can be seen in the graph, the FW at 5% increases as the set power decreases. Thus, various examples are configured with laser powers set to operate the laser in this hybrid mode to provide sufficient spot intensity for detection at the image sensor within a reasonable amount of time, but still sufficiently limit the laser power so as not to produce edge patterns that introduce unwanted instabilities in spot placement. Because lower intensities require longer exposure times for adequate readout at the image sensor, reducing the laser power can adversely affect the latency of the focus tracking system. Therefore, when determining whether sufficient intensity is provided, it may be useful to consider the amount of time required to complete the focus tracking measurement and whether the system's latency goals are adequately achieved. The amount of power applied to the laser to achieve the foregoing depends on the specified laser diode, the sensitivity and speed of the image sensor (for latency considerations), the latency requirements of the system, and the accuracy requirements of the system.

Other examples can be implemented with the laser power configured to operate the laser so that, at a given frequency, the main peak in the laser diode output has a normalized intensity that is 15%-100% greater than any secondary peaks in the laser diode output. In still other examples, the power level at which the laser diode light source is operated is selected so that, at a given frequency, the main peak in the laser diode output has a normalized intensity that is 15%-25% greater than the normalized intensity of the secondary peaks in the laser diode output. In still other examples, the power level at which the laser diode light source is operated is selected so that, at a given frequency, the main peak in the laser diode output has a normalized intensity that is 15%-100% greater than the normalized intensity of the secondary peaks in the laser diode output. In yet other examples, the power level at which the laser diode light source is operated is selected so that, at a given frequency, the main peak in the laser diode output has a normalized intensity that is 15%-200% greater than the normalized intensity of the secondary peaks in the laser diode output.

Another metric that can be used to set the power at which the light source is operated can be the maximum exposure time that the system can tolerate while meeting a predetermined focus tracking latency requirement. Generally speaking, as the power at which the laser is operated is reduced, the amount of flux at the edge of the light spot is also reduced, improving focus tracking accuracy. However, insufficient intensity is provided at the image sensor below a certain power level to enable detection of the light spot, or to enable detection with a sufficiently short exposure time to meet the latency requirement. Therefore, the power setting can be reduced to a point where the corresponding exposure time required is at or near the maximum exposure time allowed for the system latency in the focus tracking operation. In the example provided above, the exposure time for a light source operating at 0.5 mW is 250 μs.

Although various examples of the disclosed technology have been described above, it should be understood that they are presented as examples only and not in a limiting manner. Similarly, the various figures may depict example architectures or other configurations for the disclosed technology, which are completed to help understand the features and functions that may be included in the disclosed technology. The disclosed technology is not limited to the example architectures or configurations shown, but it is expected that features can be implemented using various optional architectures and configurations. In fact, it will be obvious to those skilled in the art how optional functional, logical or physical block and configuration can be implemented to achieve the desired features of the technology disclosed herein. Moreover, many different component module names other than those depicted herein can be applied to various blocks. In addition, with respect to flow charts, operational descriptions, and method claims, the order in which the steps are presented herein should not force the disclosed technology to be implemented as performing the listed functions in the same order, unless the context dictates otherwise.

Although the disclosed technology is described above in terms of various example configurations and implementations, it should be understood that the various features, aspects, and functions described in one or more individual examples are not limited in their applicability to the specific examples for which they are described, but can be applied alone or in various combinations to one or more other examples of the disclosed technology, regardless of whether such examples are described and whether such features are present as part of the described examples. Accordingly, the breadth and scope of the technology disclosed herein should not be limited by any of the above examples.

Unless otherwise expressly stated, the terms and phrases used in this document and their variations should be interpreted as open-ended and not restrictive. As an example of the foregoing: the term "including" should be understood to mean "including but not limited to," etc.; the term "example" is used to provide an example instance of the item in question, rather than an exhaustive or limiting list thereof; the term "a" or "an" should be understood to mean "at least one," "one or more," etc.; and adjectives such as "pre-existing," "traditional," "normal," "standard," "known," and terms of similar meaning should not be interpreted as limiting the items described to items available at a given time period or up to a given time, but should be understood to include pre-existing, traditional, normal, or standard technologies that may be available or known now or at any time in the future. The term "including" is defined herein as open-ended, including not only the listed elements, but also any additional elements. Similarly, where this document refers to technologies that would be obvious or known to one of ordinary skill in the art, such technologies include those that would be obvious or known to the skilled person now or at any time in the future.

The term "coupled" means to join, connect, fasten, contact or link directly or indirectly, and may refer to various forms of coupling, such as physical, optical, electrical, fluid, mechanical, chemical, magnetic, electromagnetic, optical, communicative or other coupling or combinations of the foregoing. Where one form of coupling is specified, this does not imply that other forms of coupling are excluded. For example, a component that is physically coupled to another component may refer to a (direct or indirect) physical attachment or contact between the two components, but does not exclude other forms of coupling between the components, such as, for example, a communication link (e.g., an RF or optical link) that also communicatively couples the two components. Likewise, the various terms themselves are not intended to be mutually exclusive. For example, fluid coupling, magnetic coupling, or mechanical coupling, etc. may be forms of physical coupling.

In some instances, the presence of expanded words and phrases (such as, "one or more," "at least," "but not limited to," or other similar phrases) should not be understood to mean that narrower cases are expected or required in instances where such expanded phrases may be absent. The use of the term "component" does not imply that the elements or functions described or claimed as part of the component are all configured in a common package. In fact, any or all of the various elements of the component (including structural elements) may be combined in a single package or maintained separately, and may further be distributed among multiple groups or packages.

It should be appreciated that all combinations of the foregoing concepts (provided such concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein.

The terms "substantially" and "approximately" as used throughout this disclosure (including the claims) are used to describe and account for small fluctuations, such as those due to variations in processing. For example, they may refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

In addition, the various examples described herein are described in terms of example diagrams and other illustrations. As will become apparent to one of ordinary skill in the art after reading this document, the illustrated examples and their various alternatives can be implemented without limitation to the illustrated examples. For example, the block diagrams and accompanying descriptions should not be construed as mandating a specific architecture or configuration.

Claims (7)

1. An imaging system, comprising: Laser source; A differential separation window that splits the light emitted from the light source into multiple parallel beams; A sample container includes a first layer, a second layer, and a third layer disposed on a substrate in contact with each other. These layers include a first reflective surface at the first layer of the sample container, a second reflective surface between the first layer and the second layer, a third reflective surface between the second layer and the third layer, and a fourth reflective surface between the third layer and the substrate. An objective lens is positioned to focus light from the light source onto at least one of the second and third reflective surfaces and to receive light reflected from the sample container; An image sensor including multiple pixel locations positioned to receive light reflected from the sample container; A beam splitter, wherein the beam splitter is arranged in the optical return path of the imaging system between the objective lens and the image sensor; A first blocking structure is positioned to prevent reflections from the first reflective surface from reaching the image sensor; and A second blocking structure, positioned to prevent reflections from the fourth reflective surface from reaching the image sensor, has a frame, a first elongated structural member, a second elongated structural member, and a blocking member extending from the frame. The blocking member has a blocking surface angled relative to the plane of the frame such that the blocking surface is substantially parallel to the beam splitting surface of the beam splitter. The blocking member has a triangular cross-section to reflect light out of the optical return path, through which light reflected from the sample container returns from the objective lens to the image sensor. 2. The imaging system of claim 1, wherein the second blocking structure is positioned in the optical return path to block reflections from the fourth reflective surface at the output of the beam splitter. 3. The imaging system of claim 1, wherein the frame defines an aperture, the first elongated structural member and the second elongated structural member, each elongated structural member including a first end and a second end, wherein the first elongated structural member and the second elongated structural member are connected at their first ends to opposite sides of the frame, and wherein the first elongated structural member and the second elongated structural member extend from the frame parallel to each other and in the same direction as each other; and The blocking member is an optically opaque blocking member positioned to extend between the respective second ends of the first elongated structural member and the second elongated structural member. 4. The imaging system of claim 3, wherein the second blocking structure is sized to be arranged adjacent to the beam splitter. 5. The imaging system of claim 4, wherein the width of the blocking surface of the blocking member is between 2 mm and 7 mm. 6. The imaging system of claim 4, wherein the width of the blocking surface of the blocking member is selected to block the reflected light beam from the fourth reflective surface of the sample container without interfering with the light beams reflected from the second reflective surface and the third reflective surface. 7. The imaging system of claim 3, wherein the first blocking structure includes an aperture designed to block reflected light beams from the first reflective surface of the sample container and allow light beams reflected from the second and third reflective surfaces of the sample container to pass through the aperture.